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PubMed1991

[Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors].

The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied. Monomeric forms of the plasmids can only transform B. subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid. Polymeric forms are needed for efficient transformation. Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure.

Bacillus subtilis↗

PubMed1995

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.

Amino Acid Sequence↗

PubMed2004

Novel rpoB mutations conferring rifampin resistance on Bacillus subtilis: global effects on growth, competence, sporulation, and germination.

Previously, spontaneous rifampin resistance mutations were isolated in cluster I of the rpoB gene, resulting in amino acid replacements (Q469R, H482R, H482Y, or S487L) in the Bacillus subtilis RNA polymerase beta subunit (W. L. Nicholson and H. Maughan, J. Bacteriol. 184:4936-4940, 2002). In this study, each amino acid change in the beta subunit was observed to result in its own unique spectrum of effects on growth and various developmental events, including sporulation, germination, and competence for transformation. The results thus establish the important role played by the RNA polymerase beta subunit, not only in the catalytic aspect of transcription, but also in the regulation of major developmental events in B. subtilis.

Alleles↗

PubMed1997

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation.

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Acetyl-CoA C-Acyltransferase↗

PubMed1985

Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase.

In Saccharomyces cerevisiae, the inner mitochondrial membrane protein cytochrome c oxidase is composed of nine polypeptide subunits. Six of these subunits (IV, V, VI, VII, VIIa, VIII) are encoded by the nuclear genome, and the remaining three (I, II, III) are encoded by mitochondrial DNA. We report here the existence of two nonidentical subunit V polypeptides, which are encoded by separate genes within the yeast genome. One gene, COX5a, encodes the polypeptide Va, normally found in preparations of holocytochrome c oxidase. The other gene, COX5b, encodes the polypeptide Vb, which cross-reacts with anti-subunit Va antiserum and restores respiratory competency and cytochrome oxidase activity in transformants of cox5a structural gene mutants. This polypeptide also copurifies with the holoenzyme prepared from these transformants. We have found that COX5b is expressed in vegetatively growing yeast cells, and that the Vb polypeptide can be detected in mitochondria from strain JM28, a cox5a mutant. This mutant has 15%-20% residual cytochrome oxidase activity, and it respires at 10%-15% the wild-type rate. By disrupting the COX5b gene in this strain, we show that this residual activity is directly attributable to the presence of a chromosomal copy of the COX5b gene. Taken together, these results suggest that Va or Vb can function as cytochrome oxidase subunits in yeast and that Vb may be used under some specific, as yet undefined, physiological conditions.

Base Sequence↗

PubMed1981

Assimilation of single-stranded donor deoxyribonucleic acid fragments by nucleoids of competent cultures of Bacillus subtilis.

Lysates containing folded chromosomes of competent Bacillus subtilis were prepared. The chromosomes were supercoiled, as indicated by the biphasic response of their sedimentation rates to increasing concentrations of ethidium bromide. Limited incubation of the lysates with increasing concentrations of ribonucleases resulted in a gradual decrease in the sedimentation velocity of the deoxyribonucleic acid (DNA) until finally a constant S value was reached. Incubation with sonicated, 4,5',8-trimethylpsoralen-monoadducted, denatured, homologous donor DNA molecules at 37 degrees C and concomitant irradiation with long-wave ultraviolet light of the nucleoid-containing lysates resulted in the formation of complexes of the donor DNA molecules and the recipient chromosomes. This complex formation was stimulated when nucleoids were previously (i) unfolded by ribonuclease incubation, (ii) (partially) relaxed by X irradiation, or (iii) subjected to both treatments. Monoadducts were not essential. On the other hand, the complex-forming capacity of recipient chromosomes previously cross-linked by 4,5',8-trimethylpsoralen diadducts was greatly reduced, suggesting that strand separation of the recipient molecule was involved in the formation of the complex. None of these effects has been observed when heterologous (Escherichia coli) donor DNA has been used. When the same kind of experiments were carried out at 70 degrees C, donor-recipient DNA complexes were also formed and required strand separation and homology similar to donor-recipient complex formation at 37 degrees C. However, in contrast to what was found at 37 degrees C, unfolding plus relaxation of the nucleoids, as well as the absence of monoadducts in the donor DNA fragments, resulted in a decrease in complex formation. On the basis of these results, we assume that superhelicity can promote the in vitro assimilation of single-stranded donor DNA fragments by nucleoids of competents B. subtilis cells at 70 degrees C, but that at 37 degrees C a different mechanism is involved.

Bacillus subtilis↗

PubMed1976

Influence of several nucleotides on the competence development of Bacillus subtilis.

The influence of adenosine-3',5'-cyclic monophosphate (cAMP) and other nucleotides on the competence development of Bacillus subtilis was studied. The stimulation of competence which can be achieved by exposing physiologically low-competent cells to supernatants from highly competent cultures can be inhibited with different cAMP doses. When the same cells were suspended in a minimal medium with cAMP, varying degrees of stimulatin- of competence were observed depending on the time of addition of the drug. This effect is not specific for cAMP. It appears to be correlated to an increase of the amount of DNA bound to the competent cells. cAMP activities were antagonized by equimolar doses of adenosine-triphosphate (ATP) and guanosine-triphosphate (GTP).

Adenosine Triphosphate↗

PubMed1996

Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis.

comR (pnpA) is a newly identified gene in Bacillus subtilis that is necessary for the expression of late competence genes. Transformability of a comR (pnpA) mutant is 1-5% of that seen in comR+ strains. Cloning and sequencing identified ComR as polynucleotide phosphorylase (PNPase). The PNPase amino acid sequence has 50% identity and 67% similarity with the Escherichia coli enzyme. Enzymatic assays show that this is the only PNPase activity in B. subtilis. comR (pnpA) is necessary for comG-lacZ and comK-lacZ expression, but this requirement is bypassed by a mecA disruption. In B. subtilis, the loss of PNPase has little effect on expression from a fusion of the srfA promoter directly to lacZ, but is necessary for normal expression from certain srfA-lacZ fusions that include portions of the normal srfA transcript. When a srfA-lacZ translational fusion is tested in isogenic pnpA+ and pnpA derivatives of E. coli, lower expression is seen in the pnpA mutant. Since expression from lacZ fusions to comA, sinR, and mecA appeared similar in the B. subtilis pnpA and pnpA+ strains, the loss of PNPase does not have a strong general effect on gene expression. These results suggest that PNPase may be necessary for modification of the srfA transcript in order to activate translation or stabilize the transcript, and that this may be necessary for competence development. This is the first evidence of post-transcriptional effects on the development of competence in B. subtilis.

Amino Acid Sequence↗

PubMed1993

The sequence-specific nuclear matrix binding factor F6 is a chicken GATA-like protein.

The sequence-specific DNA-binding protein factor F6, which binds upstream of the cluster of the chicken alpha-globin genes, has previously been found to interact with a DNA fragment containing a replication origin and a nuclear matrix binding site. This protein has been partially characterized. Based on its molecular weight and binding affinity, F6 belongs to a family of GATA proteins, the chicken equivalent of transcription factor NFE-1. An oligonucleotide including the binding site for F6 competes for binding of the above-mentioned DNA fragment to the nuclear matrix. This indicates an involvement of this protein in the interaction between DNA and the nuclear matrix.

Animals↗

PubMed1994

Response of repair-competent and repair-deficient Escherichia coli to three O6-substituted guanines and involvement of methyl-directed mismatch repair in the processing of O6-methylguanine residues.

Plasmids containing a site-specifically incorporated O6-methyl- (m6G), O6-ethyl- (e6G), or O6-benzylguanine (b6G) within the ATG initiation codon of the lacZ' gene were used to transform Escherichia coli that were repair proficient or deficient in one or both of the E. coli O6-alkylguanine-DNA alkyltransferases, the uvr(ABC) excision repair system, the recA-mediated recombination system, or the methylation-directed mismatch repair system. Colonies were scored phenotypically for adduct-induced mutations. With plasmids containing either e6G or b6G, the frequency of adduct-induced mutation was low and independent of the repair proficiency of the strain transformed. Plasmids containing an m6G residue elicited similar responses in all but the mismatch repair-deficient strain. The generally low mutagenicity of all the O6-substituted guanines was interpreted as reflecting an adduct-induced arrest of replication of the modified strand while the unmodified complementary strand was replicated normally. Studies of the involvement of mismatch repair in m6G mutagenesis showed that m6G:T base pairs were more readily processed than m6G:C base pairs, indicating that mismatch repair involving m6G residues occurs after replication. These data support a model in which the E. coli methylation-directed mismatch repair system diverts plasmids containing promutagenic m6G:T base pairs into replication-arrested complexes providing another line of defense against O6-methylguanine mutagenicity in addition to O6-alkylguanine-DNA alkyltransferase repair and excision repair mechanisms.

Base Sequence↗

PubMed1979

Decrease in epidermal growth factor receptor levels and production of material enhancing epidermal growth factor binding accompany the temperature-dependent changes from normal to transformed phenotype.

Normal rat kidney (NRK) cells infected with a temperature-sensitive mutant of Kirsten sarcoma virus (Ts cells) exhibited normal monolayer morphology identical to that observed for uninfected cells (NRK cells) at the nonpermissive temperature, 39 degrees C, but grew as multilayered foci resembling NRK cells transformed by the wild-type virus (KNRK cells) at 32 degrees C, the permissive temperature. NRK cell division was stimulated by epidermal growth factor (EGF), and these cells showed high levels of EGF receptors, as determined by 125I-labeled EGF binding. KNRK cells were unresponsive to EGF and no EGF receptors were detectable. Ts cells also were unresponsive to EGF at both temperatures, but exhibited just detectable EGF binding at 32 degrees C and 10-15% of NRK cell binding at 39 degrees C. Use of EGF added to the culture medium by these cells paralleled the receptor levels. Crossfeeding experiments among NRK, KNRK, and Ts cultures indicated that Ts cells at the permissive temperature and KNRK cells at both temperatures produced a heat-stable substance(s) which stimulated DNA synthesis in NRK cells independent of the presence of serum or of EGF. Conditioned medium from the transformed cultures also significantly enhanced EGF binding to NRK cells. These studies demonstrated a correlation between the transformed phenotype and the receptor levels of a potent cell mitogen, EGF, which was readily reversible in the Ts cultures. In addition, cultures expressing the transformed phenotype produced material that did not compete for the EGF receptor but did enhance EGF binding, in contrast to other reports involving sarcoma virus-transformed cells.

Animals↗

PubMed2005

[Expression of recombinant Vp6 gene of porcine rotavirus A with non-antibiotic Lactobacillus vector in Escherichia coli].

The antigenic determinants of Vp6 gene of porcine rotavirus A was amplified from infected MA 104 cell by the reverse transcription-polymerase chain reaction (RT-PCR), the product of which was a 1194bp cDNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Cloning plasmid pGEM-T-Vp6 and the prokaryotic shuttle expression vector pW425t between E. coli and Lactobacillus, were digested by SacI and KpnI double enzymes, respectively. The purified Vp6 gene was subcloned into the expression vector pW425t. Thus, the recombinant pW425t-Vp6 was constructed, which then was transformed into the competence thyA gene-mutant E. coli X13. Treated lysates of bacterium were loaded directly onto SDS-PAGE, on which approximately 44.88 kD fusion protein was observed. The protein was further analyzed using Western blot, which indicated that the protein was reactive with the antibody of rotavirus A. The results lay foundation for further studies on the Lactobacillus subunit vaccine and DNA vaccine of Vp6 gene for prevention and control of porcine rotavirus.

Animals↗

PubMed1996

[Generation of genetic engineering anti-idiotypic antibody against OC125 monoclonal antibody of ovarian carcinoma].

OBJECTIVES: To amplify and express the variable region genes of murine derived anti-idiotypic monoclonal antibody and to develop a new kind of ovarian carcinoma vaccine. METHODS: mRNA was prepared from murine hybridoma which produces anti-idiotypic antibody against OC125. The heavy and light chain variable region genes (VH and VL) were amplified by polymerase chain reaction (PCR), joined by DNA linker fragments to form single chain FV gene (ScFv), and were ligated into phage mid vector, pCANTAB 5E. The recombinant vector was induced into competent E. coli TG1 cells. The phage displayed recombinant antibodies were detected and identified by enzyme-linked immunosorbent assay (ELISA). RESULTS: The VH, VL and ScFv genes were 340 bp, 325 bp and 750 bp respectively. Some of the transformed clones expressed engineered anti-idiotypic antibodies, which could specifically bind to the original antibody (OC125). CONCLUSIONS: Genetic engineering anti-idiotypic antibody against OC125 monoclonal antibody was successfully generated. Clinical application of anti-idiotypic antibody to immunotherapy and to prevention of ovarian carcinoma may be studied.

Animals↗

PubMed1999

A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system.

The variability in gene conversion frequency by an RNA-DNA oligonucleotide (RDO) prompted us to develop a system as a means of measuring the conversion frequency rapidly and reproducibly. A shuttle vector was constructed to measure the frequency of targeted gene correction by RDO of the E. coli beta-galactosidase gene containing a single point mutation (G --> A), that resulted in inactivation of enzymatic activity. An RDO corrected the point mutation and restored the enzymatic activity, approximately 1%, determined by a histochemical staining in mammalian cells and by a color selection (blue or white) of bacteria transformed with Hirt DNA. In addition, we established an in vitro system capable of gene correction using nuclear extracts. CHO-K1 nuclear extracts corrected the point mutation approximately 0.1%, determined by bacterial transformation. Using the in vitro reaction, frequency of gene conversion in different cell types was measured. The embryonic fibroblasts from p53-/- mouse showed higher gene correction than that of the isogenic p53+/+ cells. Nuclear extracts from DT40 cells, which have a higher homologous recombination rate than any other mammalian cells exhibited 0.1-0.6% of gene correction. These results indicated that recombination may be rate-limiting in gene conversion by RDO in cells with competent mismatch repair activities. Utilizing transfection and in vitro reaction, we demonstrated that such a shuttle system might be useful in comparing the frequency of targeting among different cell types and to investigate the mechanism of gene conversion by RDO.

Animals↗

PubMed1993

Analysis of the synergistic stimulation of mouse macrophage proliferation by macrophage colony-stimulating factor (CSF-1) and tumor necrosis factor alpha (TNF-alpha).

Tumor necrosis factor alpha (TNF-alpha) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony-stimulating factor (CSF-1). However, nothing is known of how TNF-alpha affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF-alpha on CSF-1-stimulated proliferation of both primary cells (bone marrow-derived macrophages, BMMs) and a cloned growth factor-dependent macrophage cell line (S1). We show that the TNF-alpha-induced increase in [3H]TdR uptake of CSF-1-stimulated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF-alpha are direct and independent of cell number. TNF-alpha decreases the population doubling time of log-phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of S1 cells from 17 to 13 h. TNF-alpha exerts its effects on log-phase growth by increasing to the same proportion CSF-1-stimulated proliferation at all concentrations of CSF-1; that is, TNF-alpha does not shift, but rather amplifies, the CSF-1 dose-response curve. Although TNF-alpha alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF-1 greatly increases subsequent CSF-1-stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF-alpha increase CSF-1-stimulated log-phase growth and reentry of quiescent cells into the cycle equally on a molar basis (half-maximal stimulation of approximately 0.3 nM). The latter observation argues that the growth-stimulatory effects of TNF-alpha are mediated via the 55-60-kd TNF receptor. We conclude that TNF-alpha acts directly on growth-competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF-1.

Animals↗

PubMed1989

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide.

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.

Adenocarcinoma↗

PubMed2003

Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice.

Constitutively activated, tyrosine-phosphorylated signal transducer and activator of transcription (STAT) 3 plays a pivotal role in human tumor malignancy. To discover disrupters of aberrant STAT3 signaling pathways as novel anticancer drugs, we developed a phosphotyrosine STAT3 cytoblot. Using this high throughput 96-well plate assay, we identified JSI-124 (cucurbitacin I) from the National Cancer Institute Diversity Set. JSI-124 suppressed the levels of phosphotyrosine STAT3 in v-Src-transformed NIH 3T3 cells and human cancer cells potently (IC(50) value of 500 nM in the human lung adenocarcinoma A549) and rapidly (complete inhibition within 1-2 h). The suppression of phosphotyrosine STAT3 levels resulted in the inhibition of STAT3 DNA binding and STAT3-mediated but not serum response element-mediated gene transcription. JSI-124 also decreased the levels of tyrosine-phosphorylated Janus kinase (JAK) but not those of Src. JSI-124 was highly selective for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways such as those mediated by Akt, extracellular signal-regulated kinase 1/2, or c-Jun NH(2)-terminal kinase. Finally, JSI-124 (1 mg/kg/day) potently inhibited the growth in nude mice of A549 tumors, v-Src-transformed NIH 3T3 tumors, and the human breast carcinoma MDA-MB-468, all of which express high levels of constitutively activated STAT3, but it did not affect the growth of oncogenic Ras-transformed NIH 3T3 tumors that are STAT3 independent or of the human lung adenocarcinoma Calu-1, which has barely detectable levels of phosphotyrosine STAT3. JSI-124 also inhibited tumor growth and significantly increased survival of immunologically competent mice bearing murine melanoma with constitutively activated STAT3. These results give strong support for pharmacologically targeting the JAK/STAT3 signaling pathway for anticancer drug discovery.

3T3 Cells↗

PubMed1975

Competent Bacillus subtilis cultures synthesize a denatured DNA binding activity.

A protein activity has been detected in soluble extracts of competent cultures of bacillus subtilis which protects denatured DNA from nuclease solubilization. Both binding activity and competence development occur simultaneously but not coordinately, since the peak of binding activity appears 3 hr after the peak of competence. The binding activivity is not detected in physiologically noncompetent cultures, in an isogenic noncompetent cultures, in an isogenic noncompetent mutant grown through the competence regimen, or in early sporulating cells. The activity is included on Sephadex G-100 gel filtration columns but elutes in a broad nonhomogeneous peak. This binding activity may play a role in uptake of DNA and/or integration of exogenous DNA into the genome of competent B. subtilis cells.

Bacillus subtilis↗